Methamphetamine (METH) abuse and human immunodeficiency virus (HIV) infection produce a double epidemic in the United States, which is a serious global concern to public health. METH abuse and HIV infection are both recognized as major causes of neurocognitive diseases. METH use can increase the risk of acquiring HIV and enhance brain injury in the context of HIV infection. However, it is unclear about the mechanisms by which METH and/or HIV impair the central nervous system (CNS). Human brain microvascular endothelial cells (HBMEC) have been well known as main components of the blood-brain barrier (BBB) and play a key role in protecting the CNS from pathogen invasion. Recently, we identified HBMEC as an important bystander in inhibiting HIV replication in macrophages, as Toll-like receptor 3 (TLR3) signaling of HBMEC could activate IFN pathway, inducing the expression of the cellular HIV restriction factors. In the proposed studies, we will determine whether METH and/or HIV compromise TLR3 signaling-mediated immune activation of HBMEC and HBMEC-mediated anti-HIV activity. The overarching hypothesis of this R03 proposal is that METH and/or HIV impair TLR3 signaling of HBMEC, diminishing HBMEC-mediated innate immunity against HIV. We propose two specific aims: 1) to determine the effect of METH and/or HIV on TLR3 signaling of HBMEC-mediated anti-HIV activity; 2) to examine the impact of METH and/or HIV on BBB permeability in the context of TLR3 signaling of HBMEC, and whether TLR3 signaling of HBMEC has protective effect on METH and/or HIV-mediated neuronal injury. Given the high prevalence of METH use and its implication in HIV-associated dementia, the proposed studies are timely and important, which should provide insight of the role of HBMEC in BBB innate immunity in the context of METH use and/or HIV infection.